Sebastiano Grasso

A Quantitative Immunofluorescent Study of the Endocrine Cell Populations in the Developing Human Pancreas

The immunofluorescent cell content of the pancreas of 8–40-wk fetuses and of 1.5–5-mo Caucasian infants was quantitatively evaluated using anti-insulin, anti-glicentin, anti-glucagon, anti-somatostatin, and anti-pancreatic polypeptide antisera. The most significant findings are: (1) the pancreas of 8–10 wk fetuses contains a sizable population of endocrine cells reacting exclusively to anti-glicentin antiserum. This cell population decreases and disappears in later stages and is replaced by the adult type glucagon/glicentin immunoreactive cell; (2) the pancreatic polypeptide-rich region shows a lower relative endocrine cell content as compared with the glucagon-rich region and its islets appear smaller; (3) in the total pancreas, the relative (volume density) and absolute (μl) insulin cell content increases regularly with age, while the relative volume of glucagon cells peaks in fetal life (wk 17–20) to decrease in infants, although remaining at higher levels than in adults; the relative and absolute volumes of somatostatin cells are elevated in fetal and infant stages studied where they represent the second most abundant cell type, while pancreatic polypeptide cells appear to least abundant cells during prenatal and infant life. These data show several differences with the pattern of the respective endocrine cell populations in the adult pancreas.

Effect of selected hexoses, of epinephrine and of glucagon on insulin secretion in man.

Immunochemical methods of measuring serum insulin have implemented studies in man confirming in vitro data which suggest that metabolizable sugars such as mannose and glucose cause insulin secretion, whereas galactose, a nonutilizable sugar, does not. Infusions of 2-deoxy-D-glucose are associated with greatly increased epinephrine secretion and sustained hyperglycemia, but no rise in serum insulin levels. Epinephrine infusions induce and prolong hyperglycemia without raising serum insulin levels and effectively inhibit insulin secretion during glucose administration. These findings agree with in vitro evidence that epinephrine directly inhibits pancreatic secretion of insulin. Glucagon, on the other hand, sharply stimulates insulin secretion, either alone or after glucose infusions. Data are presented which suggest that this may be a direct effect of glucagon on the release of insulin by the pancreatic beta cell.

Placental thrombosis and fetal loss after passive transfer of mouse lupus monoclonal or human polyclonal anti-cardiolipin antibodies in pregnant naive BALB/c mice.

In the present study we evaluated the effect of passive transfer of a mouse monoclonal (CAM) or a human polyclonal anti‐cardiolipin IgG on pregnancy outcome in BALB/c mice. The mice were immunized through the tail vein immediately after mating with 10 μg of monoclonal or polyclonal anti‐cardiolipin antibodies. Two other groups of mice were given a mouse irrelevant monoclonal antibody or normal human polyclonal IgG respectively, at the same dose. In mice immunized with monoclonal or polyclonal anti‐cardiolipin antibody we observed a significant increase in the number of fetal resorptions and a significant reduction of the mean weights of the embryos and the placentas. In mice immunized with CAM we also found a significant decrease in the number of healthy pups, while mice infused with human aCL antibody expressed a significant reduction in the fecundity rate. The histological examination showed widespread thrombosis and necrosis in the placentas derived from the mice immunized with the anti‐cardiolipin antibodies. The model supports a possible direct pathogenetic effect of anti‐phospholipid antibodies in recurrent fetal loss and points out that thrombotic events at placental level can be instrumental in the pathogenesis of the obstetric complications.

Prevention of Spontaneous Autoimmune Diabetes in Diabetes-Prone BB Rats by Prophylactic Treatment with Antirat Interferon-γ Antibody

The role of endogenous interferon-γ (IFNγ) in the development of insulin-dependent diabetes mellitus (IDDM) in diabetes-prone BB rats was evaluated. Several groups of these animals were treated under different experimental conditions with a purified polyclonal antibody (Ab), antirat IFNγ. The results show that when administered at doses of 100 or 200 μg/week from the 30/33th until the 105th day of age, the anti-IFNγ Ab reversibly reduced the incidence of IDDM compared to that in control rats treated with either irrelevant rabbit IgG or PBS. Moreover, when given up to the 105th day of age, these doses of anti-IFNγ Abs exerted comparable preventive effects regardless of whether application started as early as within 24 h after birth or at the end of the prediabetic period (e.g. 70/75 days). In contrast, under none of the above experimental conditions did larger doses of anti-IFNγ Ab (500 μg or 1 mg/week) exert antidiabetogenic effects in the BB rats. Apparently, this was due to the exuberant production of n...

Dichotomic effects of IFN-γ on the development of systemic lupus erythematosus-like syndrome in MRL-lpr/lpr mice

Systemic lupus erythematosus (SLE)‐prone female MRL‐lpr / lpr (MRL‐lpr) mice were treated with mouse or rat IFN‐γ under different experimental conditions, both prophylactically in 6‐ to 8 week‐old animals and therapeutically in 12‐ to 18‐week‐old SLE‐affected mice. It was found that IFN‐γ heterogeneously modulated the course of the disease in MRL‐lpr mice. When administered prophylactically, IFN‐γ favorably modulated the histological, serological and clinical signs of the disease. Relative to untreated or PBS‐treated control animals, the MRL‐lpr mice which received IFN γ were virtually free of inflammatory infiltration of the kidneys and the lungs, had lower levels of azotemia with reduction of both circulating IgG1, IgG2a and IgG3 and anti‐double strand (ds) and single strand (ss) DNA antibodies, milder skin vasculitis, significantly reduced enlargment of their lymph nodes and lower weight of the spleens. IFN‐γ also lowered the rate of mortality of MRL‐lpr mice. In contrast to these findings, therapeutically administered IFN‐γ worsened the course of the disease in MRL‐lpr mice, which exhibited increased proteinuria, higher levels of IgG2a and IgG3 and anti‐ds and ‐ss DNA antibodies, more aggressive nephritis and died at an earlier age than PBS‐treated control mice. The dichotomic effect of IFN‐γ on disease manifestation in MRL‐lpr mice offers new insights into the complex role of this cytokine in the regulation of systemic autoimmunity such as SLE.

Insulin Secretion in the Premature Infant: Response to Glucose and Amino Acids

Serum insulin was measured in fifty-one premature infants in the first twenty-four hours of life following the administration of glucose, a mixture of amino acids, or glucose with an amino acid mixture. Infusion of glucose (1.25 gm.) caused a rapid increase of blood glucose and a small rise in serum insulin. The amino acid mixture (2.5 gm.) produced, on the contrary, a rapid and marked increase of serum insulin but only a slight rise of blood glucose. When the dose of the amino acid mixture was halved, rise of serum insulin and blood glucose was small. Combining the reduced dose of amino acid mixture with glucose produced a rapid and striking increase of both serum insulin and blood glucose.

Paradoxical Antidiabetogenic Effect of γ-Interferon in DP-BB Rats

Previous studies have shown that anti-γ-interferon (IFN-γ) antibody reduces the frequency of autoimmune IDDM in the DP-BB rat. We tested the effects of systemically administered recombinant rat IFN-γ in both DP-BB and DR-BB rats. Unexpectedly, IFN-γ markedly reduced the incidence of IDDM as compared with control rats when administered six times per week at a dosage of 280,000 U between ages 30-35 to 105 days or ages 60-64 to 105 days. A lower dosage (28,000 U on alternate days) was also protective when administered to DP-BB rats between birth and age 60 days. However, long-lasting protection against IDDM development over the 1-year study period was achieved only by the highest dosage of IFN-γ administered from age 30 to 105 days. Ex vivo production of tumor necrosis factor-a from splenic lymphoid cells (SLCs) and peritoneal macrophages of the rats treated with IFN-γ was comparable with that of controls; however, SLCs from the IFN-γ-treated animals secreted lower amounts of IFN-γ after stimulation with concanavalin A. IFN-γ treatment also markedly reduced the frequency of phenotypically activated SLC-expressing class II antigens and interleukin-2 receptor. Finally, in agreement with the observed antidiabetogenic effects, exogenously administered IFN-γ induced neither insulitis nor IDDM development in DR-BB rats, a subline of DP-BB rats in which autoimmune diabetes rarely occurs spontaneously but can be induced by administration of polyinosinic-polycytidilic acid.

The pancreatic polypeptide-rich lobe of the human pancreas: Definitive identification of its derivation from the ventral pancreatic primordium

Sir, The human pancreas, in common with that of most mammals studied so far, shows a very uneven distribution of two of the endocrine cell types, the glucagonand the pancreatic polypeptide containing cells. Pancreatic polypeptide containing cells are concentrated in a posterior region of the pancreatic head which is extremely poor in glucagon cells [1]. This pancreatic polypeptide rich region often constitutes an individual lobe at the posterior and inferior pole of the head which can be cleaved along a connective-vascular plane from the remainder of the pancreas [2]. The embryological development of the pancreas, during which two distinct primordia merge to yield the definitive organ [3], suggested that the pancreatic polypeptide rich region has its origin in the ventral primordium [41. Although this hypothesis was supported by studies of fetal and neonatal pancreas [5], direct proof has remained elusive. This is because pancreatic polypeptide containing cells are undetectable by immunocytochemical methods in the very early stages of development when the ventral and dorsal primordia are unfused. Pancreatic polypeptide containing cells become evident from week 10 of gestation onwards [6], at a stage when all trace of fusion has disappeared. By the systematic examination of the pancreas at autopsy from fetuses of various ages, we have discovered an annular pancreas which provides what we believe to be proof that the pancreatic polypeptide rich region has its origin in the ventral primordium. Pancreases from legally aborted fetuses were fixed in toto in Bouin solution for 24 h, cut in three parts (head, body and tail) and separately dehydrated and embedded in paraffin. An annular pancreas was found in an 11-cm fetus, aborted during total hysterectomy for uterine leiomyoma. Horizontal paraffin sections of the head stained with hemalum-eosin confirmed that the duodenum was entirely surrounded by pancreatic tissue (Fig. 1A). Unstained sections (5 gm thick) were incubated with anti-bovine pancreatic polypeptide antiserum (dilution 1/200, a gift from Dr. R.Chance, Indianapolis). By using the indirect immunofluorescence method [7] to reveal the binding sites of the antiserum, it was possible to show immunofluorescent cells (Fig. 1B) in four-fifths of the circumference of the annular pancreatic tissue, but in none of the remaining one-fifth. In contrast, sections of the latter part showed numerous immunofluorescent cells (Fig. 1C) following incubation with an anti-glucagon antiserum (dilution 1/200 , a gift from Dr. R. H. Unger, Dallas). On at least one side of the annular pancreas, the zone of transition between pancreatic polypeptide rich and poor regions was marked by an uninterrupted plane

Protection from concanavalin A (Con A)‐induced T cell‐dependent hepatic lesions and modulation of cytokine release in mice by sodium fusidate

The immunomodulatory effects of the antibiotic sodium fusidate (SF) were tested in a model of T cell‐dependent hepatic injury that can be induced in normal mice by a single i.v. injection of Con A. Signs of hepatitis with elevated transaminase activities in plasma, severe infiltration of the liver by neutrophil granulocytes, lymphocytes and monocytes, and necrotic areas were observed in control mice treated intraperitoneally with PBS 24 h and 1 h before Con A challenge. T cell‐ and macrophage‐derived cytokines (IL‐2, interferon‐gamma (IFN‐γ), tumour necrosis factor‐alpha (TNF‐α), IL‐1β, IL‐6) were released with different kinetics in the circulation of these mice. SF, 20, 40 or 80 mg/kg, administered 24 h and 1 h before Con A challenge, protected the mice against the hepatitic effects of Con A. The protective effects of SF were dose‐dependent and accompanied by profound modifications of blood levels of cytokines induced by Con A, so that, relative to control mice, SF (80 mg/kg)‐treated animals showed markedly diminished plasma levels of IL‐2, IFN‐γ and TNF‐α, along with augmented levels of IL‐6. These results suggest that SF might be useful in the treatment of immunoinflammatory liver diseases in humans.

In vivo treatment with a monoclonal antibody to interferon-gamma neither affects the survival nor the incidence of lupus-nephritis in the MRL/lpr-lpr mouse

The effects of the in vitro treatment with a mAb (DB-1) that neutralizes mouse IFN-gamma on the development of the SLE-like syndrome in MRL/lpr-lpr (MRL-lpr) mice were studied. The results show that the i.p. administration of 2.6 mg/week of DB-1 from the 12th to the 20th week of age neither affected the survival nor the incidence and severity of lupus nephritis in MRL-lpr mice. This study argues against the pathogenic relevance of IFN-gamma in this experimental model of human SLE.